Previous work by others has described the preparation of gold and silver colloids. Such colloids do not have a fixed number of metal atoms and vary considerably in size. For example, the metal colloids could vary in size from 1 nm to 2 .mu.m in size and could contain from about 10 metal atoms to thousands of metal atoms, depending on size. It was found that a number of proteins, such as IgG antibodies, could be adsorbed to these sol particles. Gold colloids have been most commonly described. These conjugates have been used in electron and light microscopy as well as on immunodot blots for detection of target molecules. These conjugates have many shortcomings. Since the molecules are only adsorbed onto the colloids, they also desorb to varying extents. This leads to free antibody which competes for antigen sites and lowers targeting of gold. Furthermore, the shelf life of the conjugates is compromised by this problem. The `sticky` colloids also tend to aggregate. If fluorescence is used to detect the target molecules, the gold particles quench most of it. Also, the gold colloids must be stabilized against dramatic aggregation or `flocculation` when salts are added by adsorbing bulky proteins, such as bovine serum albumin. Due to the effects of aggregation and bulky additives, the penetration of immunoprobes into tissues is generally&lt;0.5 .mu.m. Accessibility of the probes to internal cell structures, e.g., nuclear proteins, or to cells deeper in a tissue sample, is impeded by these properties. Colloidal gold immunoprobes are also used in diagnosis on blots. The sensitivity of these detection schemes is again degraded by problems of aggregation, detachment of antibodies from the gold, and problems with shelf life. The gold prepared in standard ways also has low activity due to few adsorbed antibodies and denaturation of some antibodies during adsorption.
Various metal cluster containing organic shells have also been previously described, such as Au.sub.11 Ph.sub.7 (Ph=phenyl), Au.sub.25 R (R=organic), Pd.sub.561, and others. These metal clusters have a fixed number of metal atoms in their metal cores which range in size from ca. 0.7-2.2 nm. Most of these metal clusters are based upon reduction of metal-triphenyl phosphines or the use of 1,10-phenanthroline.
For example, Barlett, P. A. et al, in "Synthesis of Water-Soluble Undecagold Cluster Compounds . . . ," J. Am. Chem. Soc., 100, 5085 (1978), describe a metal cluster compound (Au.sub.11) having a core of 11 gold atoms with a diameter of 0.8 nm. The metal core of 11 gold atoms in the undecagold metal cluster compound is surrounded by an organic shell of PAr.sub.3 groups. This metal cluster compound has been used to form gold immunoprobes, for example, by conjugating Au.sub.11 to Fab' antibody fragments as well as other biological compounds.
Another metal cluster compound which has been used as a probe is Nanogold.TM. available from the assignee of the present application. Nanogold.TM. has a metal core with 50-70 gold atoms (the exact number not yet being known but believed to be 67 gold atoms) surrounded by a similar shell of organic groups (PAr.sub.3) as undecagold. The metal core of Nanogold.TM. is 1.4 nm in diameter. The production of Nanogold is described in pending application Ser. No. 988,338, filed Dec. 9, 1992, of James F. Hainfeld and Frederic R. Furuya.
Although the preparation and properties vary for these metal cluster compounds having organic shells, many of these can only be synthesized in low yields, derivatization for use in coupling to biomolecules is expensive in time and effort, and again in low yields, and many of the cluster compounds are degraded rapidly by heat or various chemical reagents.